Deubiquitinating enzymes play critical regulatory roles in the ubiquitin-proteasome pathway (UPP). Despite their relevance to human disease and interest by pharma as targets for therapeutic development, this diverse class of enzymes is understudied and detailed biochemical data is lacking. Highly purified DUB enzymes and appropriate substrates for sensitive and homogeneous assays are not currently commercially available. In Phase I we made new ubiquitin (Ub) proteins and poly-ubiquitin chains which are successful DUB substrates. This project was successful and generated a comprehensive tool set with wide-ranging that facilitated the identification and characterization of new DUBs and other UPP components. In this proposal we build on that progress and focus on two major topics: new deubiquitinating enzymes and novel ubiquitin chain substrates. Our specific aims are to: i) develop new DUBs to expand our current range of commercially available enzymes; ii) test the complete set of DUBs against the Ub substrates and chains made in Phase I; and iii) use new modification on Ub chains to develop a continuous flour-based assay system for DUB proteolysis. We will generate the most comprehensive panel of such proteins that are commercially available. We will characterize these DUBs using our existing Phase I generated substrates to understand their biology and possibly shed light on their function in the cell. The Phase I poly-ubiquitin chain prototypes will be the starting point for modified chains, creating a universal DUB substrate that is optimal for fluorescent-based assay formats such as fluorescent resonance energy transfer (FRET). The substrate will also contain relevant isopeptide bond linkages at lysine positions 48 and 63, in order to dissect DUB linkage preferences. These reagents will be commercialized to further advance DUB research and drug discovery in protein catabolism, signal transduction, cell cycle, apoptosis, and neurodegeneration. [unreadable] [unreadable] PUBLIC HEALTH RELEVANCE: SBIR research project is to support the design, development, and production of biological products and diagnostic laboratory analysis kits. Specifically it relates to the molecular biochemistry of the human protein "ubiquitin" which has roles in numerous disease states such as cancer, inflammation, infection and neurodegeneration. The research data and useful kit products generated by this project will enable new research and drug development in the ubiquitin pathway for various human diseases. [unreadable] [unreadable] [unreadable]